Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities

Abstract

Camelina sativa is an emerging biotechnology oil crop. However, more information is needed regarding its innate lipid enzyme specificities. We have therefore characterized several triacylglycerol (TAG) producing enzymes by measuring in vitro substrate specificities using different combinations of acyl-acceptors (diacylglycerol, DAG) and donors. Specifically, C. sativa acyl-CoA:diacylglycerol acyltransferase (DGAT) 1 and 2 (which both use acyl-CoA as acyl donor) and phospholipid:diacylglycerol acyltransferase (PDAT, with phosphatidylcoline as acyl donor) were studied. The results show that the DGAT1 and DGAT2 specificities are complementary, with DGAT2 exhibiting a high specificity for acyl acceptors containing only polyunsaturated fatty acids (FAs), whereas DGAT1 prefers acyl donors with saturated and monounsaturated FAs. Furthermore, the combination of substrates that resulted in the highest activity for DGAT2, but very low activity for DGAT1, corresponds to TAG species previously shown to increase in C. sativa seeds with downregulated DGAT1. Similarly, the combinations of substrates that gave the highest PDAT1 activity were also those that produce the two TAG species (54:7 and 54:8 TAG) with the highest increase in PDAT overexpressing C. sativa seeds. Thus, the in vitro data correlate well with the changes in the overall fatty acid profile and TAG species in C. sativa seeds with altered DGAT1 and PDAT activity. Additionally, in vitro studies of C. sativa phosphatidycholine:diacylglycerol cholinephosphotransferase (PDCT), another activity involved in TAG biosynthesis, revealed that PDCT accepts substrates with different desaturation levels. Furthermore, PDCT was unable to use DAG with ricineoleyl groups, and the presence of this substrate also inhibited PDCT from using other DAG-moieties. This gives insights relating to previous in vivo studies regarding this enzyme.

Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil

“…Recent studies using enzyme assays suggest that different DGAT families have different specificities toward acyl-donor and acyl-acceptor. Camelina CsDGAT1 excludes, whereas CsDGAT2 selects, acyl-acceptors containing only polyunsaturated FAs [ 100 ]. In Brassica napus , DGAT1 variants prefer acyl-donors with saturated (e.g., 16:0-CoA) and monounsaturated (e.g., 18:1-CoA) acyl-donors, whereas DGAT2 variants strongly select 18:3-CoA [ 101 ].…”

“…A mutation in CsPDAT1 reduces oil content [ 118 ], and either up-regulating or down-regulating CsPDAT1 does not change seed oil content [ 118 , 119 ]. Studies indicate that CsPDAT1 has a substrate preference for polyunsaturated FAs, 18:2 and 18:3 [ 100 , 117 , 118 , 119 ]. Similar results are also reported for AtPDAT1 [ 111 ], sunflower PDAT1 [ 120 ] and flax PDATs ( LuPDAT1 and LuPDAT5 ) [ 121 ], with the exception of sesame ( Sesamum indicum ), where the over-expression of SiPDAT1 in yeast H1246 results in increased 18:1 and 18:2 levels, but not 18:3 [ 122 ].…”

“…The PlPDCT protein sequence shared 73.1–86.4% with AtPDCT and CsPDCTs, and 61.8% with RcPDCT ( Supplementary File 6, Table S18 , Figure 13 B). AtPDCT [ 41 ] and CsPDCT [ 100 ] are not able to use HFAs substrates, whereas in species where the seed oils contain high levels of unusual FAs, such as 18:1OH in castor and cyclopropane FAs (CPAs) in lychee ( Litchi chinensis ), their PDCTs have been shown to facilitate the efficient accumulation of HFAs in Arabidopsis [ 41 ] and CPAs in Camelina [ 127 ]. The alignment of the PlPDCT with AtPDCT, CsPDCT, and RcPDCT revealed a central core of high aa similarity ( Figure 13 C), showing a catalytic triad with a set of three coordinated amino acids, H-H-D (or His-His-Asp), in the conserved C2 and C3 domains of the lipid phosphatase/phosphotransferase family [ 128 ].…”

LPEATs Tailor Plant Phospholipid Composition through Adjusting Substrate Preferences to Temperature

“…The VLCFA are the dominant pool of acyl-CoA profile of C. sativa seeds [ 44 , 48 ]. Nevertheless, enzymes’ preference towards them was detected on a negligible level assuming that they do not participate in G3P pathway leading to TAG synthesis in C. sativa seeds [ 48 , 53 ]. TAG synthesis supplementation via LPCAT activity, which may introduce fatty acids into the PC pool was examined in a study conducted by Klińska et al [ 28 ], where LPCAT enzymes present in C. sativa seeds did not exhibit any activity, in forward reaction, towards these acyl donors.…”

Camelina sativa phosphatidylcholine:diacylglycerol cholinephosphotransferase‐catalyzed interconversion does not discriminate between substrates

“…The activity of PDCT plays a central role in channeling polyunsaturated acyl groups from PtdCho to TAG via DAG in oilseeds such as soybean (Bates et al, 2009) and Arabidopsis (Bates & Browse, 2012;Lu et al, 2009). A few reports have demonstrated in vitro activities of the PDCT enzyme by assaying it in microsomal fractions from yeast expressing the enzyme (Bai et al, 2020;Hu et al, 2012;Lager et al, 2020;Lu et al, 2009) Camelina PDCT has a strict requirement for PtdCho and sn-1,2-DAG as substrates…”